Process for obtaining provitamin d



Patented Aug. 1946 PROCESS FOR OBTAINING PROVITAMIN D dstown, N. J., and George William S. Calcott, Woo

E. Holbrook and Stockton G.

Turnbull, J r.,

Wilmington, Del., assignors to E. I. du Pont de Nemours & Company, poration of Delaware Wilmington, Del., a cor- No Drawing. Application July 11, 1942,

Serial No. 450,632

This invention relates to a new and improved method for obtaining fat-soluble compounds and more particularly refers to a process for separating provitamin D from animal organisms containing the same.

Heretofore fat-soluble substances generally,

and in particular provitamin D, were obtained from animal organisms by treating them with a saponifying agent which was generally an alcohol-water solution-oi caustic soda. Where the animal organism was enclosed in a protective shell as in the case of shellfish, it was first necessary to remove this shell before subjecting its contents to the foregoing saponification treat ment. Needless to say, this process was tedious and expensive.

A further disadvantage of the prior art was the 7 Claims. (Cl. 167-81) treatment of' mussels and other shellfish with a saponifying solution for a sufiicient period of time and under such conditions as to release the provitamin D content the'reof into the saponifying liquor, and the reuse of such liquor in the extraction of provitamin D from additional mussels or shellfish. In a still more restricted sense this invention isconcemed with a process whereby mussels or other shellfish are crushed, the crushed mass treated with a saponifying solution, the shells and other insoluble constituents thereafter removed from the reaction zone and additional quantities of crushed mussels or shellfish introduced into said reaction zone for a similar treatment. The process is continued until the concentratlon of provitamin D in the saponifying liquor necessity of employing extremely large quantities of saponii'ying solution for the recovery of a small amount of provitamin D. This, too, resulted in a great increase in the cost of the process because of increased handling charges, the necessity of using extraordinarily large amounts of organic solvents for recovering the provitamin D from the saponifying solution and the loss of appreciable amounts of these expensive agents.

It is an object of the present invention to overcome the aforesaid disadvantages and other disadvantages directly or indirectly resulting therefrom. A further object is to obtain fat-soluble constituents from'animal organisms containing the same in a simple and more economical manner than was heretofore possible. A still further object is to obtain solution of provitamin D from shellfish by means of a process wherein the expensive removal of the shell is avoided, the use of alcohol is unnecessary and the amount of chemical agents necessary is greatly reduced. I A still further object is to obtain solutions of provitamin D from mussels and other shellfish in a I more concentrated and cheaper manner than was heretofore possible. Additional objects will become apparent, from a consideration of the following specification and claims.

. These objects are attained in accordance with the present invention wherein fat-soluble con-' stituents are removed from animal organisms by is suificiently high to render its subsequent handling and extraction with organic solvent economically feasible. In its preferred embodiment this invention is concerned with the treatment of mussels to obtain therefrom their provitamin D content by washing, steaming and draining the mussels, crushing the shells thereof and subjecting the resulting mass of cracked shell and meat to the action of a saponifying solution of aqueous sodium hydroxide solution for a suflicient period of time and under such conditions as to release substantially all the provitamin D, removing the shell from said solution and repeating the proc-'- ess with additional mussels, treated a aforesaid, and the original saponifying solution until the liquor becomes highly concentrated in provitamin D.

The invention may be more readily understood by a consideration of the following illustrative examples: v

' EXAMPLE 1 Production of provitamin D from the mussel,

' Modiolu demissus (Dillwyn) or crushed) from 30.0 parts of whole mussels of treating said organisms in the presence of such protective coatings asthey may possess with a saponifying solution, removing the protective coating from the reaction zone and repeating theprocess with additional organisms but the original saponiiying liquor until a concentrated liquor oi rat-soluble constituents is obtained. In a more restricted sense this invention pertains to the the species, Modiolus demissus (Dillwyn). The temperature of the caustic was kept at '95-97" C. for one-half hour, and then the basket, which now contained nothing but shells, was raised from the vessel. The caustic liquor was allowed to drain back into the saponiflcation vessel. The basket and shells were then washed by immersion I Production of ,;then put into jfiake caustic was added to give a lution, and thirty 1 oily solid, which was dissolved in ten 1 alcohol.

tals. These sterols may To the caustic liquor in the saponification vessel there was added 0.50 part of so d flake caustic, and another saponification operation was performed on the crackings" from 30.0 parts of whole mussels. The shells after being stripped of the meat'were washed in the same wash water used after the previous saponification. In the same manner, using the same caustic liquors and same wash water, throughout, the meat was re' moved from thirty batches of crackings," each from 30.0 parts of whole mussels. After the last batch of. crackings had been processed in this manner, the caustic liquor in the saponification vessel was held at 95-97 C. for four hours and was then withdrawn. The wash water liquor was the saponification vessel, sufllcient caustic somore batches of crackings from 30 parts of whole mussels, were consecutively processed as outlined above.

The saponification liquor, after being heated at 95-97" C. was twice extracted with an appropriate solvent such as ethyl ether, in the proportion of 3 parts The combined ether extracts were washed .with

of caustic liquor to one part of ether.

water and the ether was removed by distillation. The unsaponiiiable matter parts of hot On cooling, the sterols containing the provitamin D crystallized in beautiful white crys be further purified by recrystallization, decolorization with charcoal or animal black, conversion to esters, followed by subsequent saponification, chromatographic adsorption, etc. An essentially quantitative yield (based on analytical methods of assay) of provitamin Dis obtained by this procedure.

Exnlrts 2 provitamin D from the periwinkle,

La'ttori a littorea By the method outlined in Example 1, batches of crackings, each from parts of the live univalve, Littorina litton-ea, were stripped of the meat in 16 parts of 15% caustic. The saponification liquor was then extracted with ether, which on concentration gave crude sterols.

These after one recrystallization from alcohol gave almost colorless sterols with a 14% provitamin D content determined by spectrographic assay.

EXAMPLE 3 Provitamin D from the oyster drill,

Urosalpinx cinereus Exllmrts 4 Provitamin D from the mussel, Mytz'lus hamatus,

The crackings from 20 batches of 30.0 parts each of these whole mussels, which are also known as the hooked mussel, were processed by the method given in Example 1. The sterols conwas obtained as an i tions hereof. V It is advisable to first clean the shellfish orothen; may bedon, by

taining the provitamin D were isolated in almost quantitative yield by crystallization from alcohol.

scribed in the previous examples to obtain almost all of the provitamin D originally present mixed with other sterols.

I Exmrns 6 Prwitamin D from fresh water mussels These mussels, which are all Lamellibranchia, are of the family Unionidae, whichcomprises 60 or more genera, among which may be listed the Quadrula, Pleurobema, Unis, Anodonta, Lampsilis, Tritigom'a, Cyzilas, Thepalia, etc. These are known in various locations a niggerheads, sand shells, pig toes, maple leaves, buckhorns, washboards, three ridges, pink elephant ears, pocketbooks, etc.

The crackings" from ten batches of 30.0 parts each of a mixture of these fresh water mussels were treated as described previously, and the provitamin D and other sterols were isolated in the usual manner.

EXAMPLE 7 Prmn'tamin D from the crab, Cancer pagwrus 'The "crackings from ten batches of 50.0 parts,

each of the steamed crabs were exposed to the action of the same 15% caustic at -l00 C. for one hour p'er batch. The caustic liquors, when processed as described previously, yielded the sterols. It is to be understood that the foregoing examples are illustrative merely of a modifications of which this invention is capable. They may be varied widely both with respect to the individual reactants and the condition of reaction without departing from the scope of this invention.-

In place of the mussels and other shellfish referred to in the examples, or in addition thereto,

any substance containing fat-soluble constituents may be employed; in particular, any substance containing provitamin D. Asa general rule it may be stated that the use of shellfish is more desirable for the production of the largest quantity of provitamin D at the most reasonable cost. Among the many types of shellfish which are contemplated for use herein, in addition to those referred to in the examples, mention may be made of oysters, clams, shrim oysters, Japanese oysters," and the like.

scallops, coon While the use of shellfish gener-- ally, and musselsv particularly is-preferred,,it contemplated that other bony animals -or,,fish=,

may be processed in accordance with the instru sources or provitamin D; this washing them thoroughly. ,Then ,theishellfish may be treated with livesteam or hotawater at atmospheric or superatmospheric pressure: steaming treatment is particularly helpful when it is desired torelax the muscles holding the shells together. Th water which is bound in the animal may then be drained out in order to prevent undue dilution of the saponifying solution to be subsequently used. At this stage the mussel or other shellfish maybe cracked in order to rup-v s its shell and permit more intimate contact few of the many sea snails, lobsters dine, etc. As strong'acids may be injurious aforesai steps may be hanged my be omitted entirely,

a1 may be steamed and cracked open prior to washing. likewise, the

e earth groups; with weak acids such carbonate, etc.; aqueous solutions 01' orsaniebases such as tetramethyl ammonium hydroxide, pyrito the fat-soluble constituents, their use is not ordinarily advisable.

may be varied widely in: the desirable results. As

ble amount of provitamin D.

When the provitamin D and/or other tat-soluble constituents are released by the saponi'iying solution the shells, it any, and/or other insoluble constituents should be action zone.

The operation may then be repeated using a fresh supply of provitamin-D-containing materials or of materials containing other fat-soluble this solution now than'ior a single operation. en the saponiiication liquor contains the desired amount or provitamin D and/or other rat- 'and some 01' them 1 if desired. I'br instance.

as sodium acetate, sodium in: this reaction to completion maybe relied upon, such as for example, auperatmospheric pressures. etc. I When the saponiflcation reaction is completed or brought to the deaired degree of partia mwith pletion the provitamin constituents may be removed therefrom in known derstood that numerous other binations thereof may likewise be used.

This latter extraction operation In the same manner the process may be modifed by introducing iine streams oi warm. benmm below the surface of the warm saponiflcation liquor.

The rat-soluble constituents obtained in the foregoing manner, D. may be isolated and purified,

animal black, conversion to esters, followed by subsequent saponification, chromatographic adsorption and the like.

By means of the present invention prior art processes for the recovery oi other fat-soluble material are greatly simplified group of materials able at an appreciable savings in cost. Likewise, numerous sources of these valuable materials which formerly were unavailable because of the prohibitive cost of treatment, are now made commercially feasible.

As many apparently widelydiiferent embodiments of this defined in the appended-claims:

We claim:

1. A process for obtaining provitamin D rrom shellfish which comprises treating said shellfish D or other fab-soluble a saponifying solution remove therefrom,

substantially tamin D, separating the tying liquor and Ztraction of material lirom additional mussels until jiution of provitaminD is obtained.

mussels which comprises sels, after they 1 drained, with a saponifying solution to remove 5 therefrom substantially all the material containing provitamin'D, separating asoensa material containing provitamin shells from the .saponii'yingliquor and reusing said liquor for-the extraction or taming provitamin D from additonal' shellfish. i 2. A process for obtaining provitamin D I shellfish which comprises treatingsaid shellfish with a saponifying solution to remove therefrom material containing provitamin D, separating the shells from the saponifying liquor and reusing said. liquor tor'the extraction of material containing provitamin D from additional shellfish until a'concentrated solution of provitamin D is obtained. a

h 3. A process for obtaining provitamin D from mussels which comprises treating said mussels with a saponiiying solution to remove therefrom reusing said liquor for the excontaining provitamin D a concentrated so- D, separating the material confrom additional mussels.

p niiyi e liquor and reusing said liquor for the extraction of material containing 5. A process for obtaining provitamin D from mussels which comprises treating crackedmussels, after they have been washed, steamed and drained, with a saponifying solution of aqueous tially all the material containing provitamin D,

all the material containing provishells from the saponi 4. A process for obtaining provitamin D from treating cracked mushave been washed, steamed and the shells from the saseparating the shells and reusing said liquor for the extraction or material containing provitamin D from additional mussels until a concentrated solution of provitamin D is obtained. 7

6. A process for obtaining provitamin D from mussels which comprises, treating cracked mussels with a saponiiyingsolution of aqueous sodium hydroxide to remove therefrom material containing *provitamin D, and separating the shells from the saponiiying liquor. 7. In a process of obtaining the unsaponifiable fraction from shell fish the step which comprises treating the whole broken open shell fish with caustic alkali stituents and liqueiy the shell fish meat.

WILLIAM S. CALCOT'I. GEORGE E. HOLBROOK. STOCKTON G. TURNBULL, JR.

provitamin D from the saponiiying liquor to saponify the saponifiable con 

